Strategy for Analyzing Limited Samples

When limited sample availability restricts the number of possible real-time PCR reactions, TaqMan® PreAmp Master Mix components enable experiments that would otherwise be impossible. Although preamplification of cDNA generated from purified RNA has been successful, the labor and time required to prepare RNA samples can be significant. The new TaqMan PreAmp Cells-to-CT™ Kit combines the convenient Cells-to-CT lysis and reverse transcription (RT) reagents with TaqMan PreAmp Master Mix components. The result is an extremely user friendly and high throughput solution for sample-limited experiments involving cultured mammalian cells (Figure 1).

Figure 1. TaqMan® PreAmp Cells-to-CT™ Kit Workflow. The TaqMan PreAmp Cells-to-CT Kit includes all the reagents to lyse, reverse transcribe, preamplify, and perform qPCR on limited samples. Users only need to supply the cultured cells and primers/probe for real-time PCR.

The use of cell lysates from the TaqMan PreAmp Cells-to-CT Kit resulted in expression profiles that are equivalent to those obtained from purified RNA. In the experiment described in this article, a pool of 100 assays for preamplification was used to demonstrate the robustness of the preamplification step over a wide variety of assays (Figure 2). Further, a range of 10–100,000 cells used with the TaqMan PreAmp Cells-to-CT Kit showed consistent and unbiased preamplification, regardless of cellular input, and superior sensitivity compared to purified RNA, particularly at low cell numbers (Figure 3).

Figure 2. CT Values Before and After Preamplification Are Not Biased by Cells-to-CT™ Lysates. HeLa cells (1 x 104) were lysed using TaqMan® PreAmp Cells-to-CT Kit or subjected to RNA purification. After reverse transcription, samples were analyzed for 100 targets with, or without, multiplexed preamplification. For real-time analysis, reactions were performed in triplicate. (A) This graph shows raw CT values from purified RNA (X-axis) and Cells-to-CT lysates (Y-axis). (B) This graph shows raw CT values after preamplification using the same cDNA as in panel A.

Figure 3. Superior Performance of Cells-to-CT™ Lysates Versus Purified RNA Over an Input Range of 10-100,000 Cells. HeLa cells were lysed using TaqMan® PreAmp Cells-to-CT Kit or RNA was purified (Supplier Q). Reverse transcription and preamplification were performed using supplied reagents. Real-time PCR was performed on both the cDNA and diluted preamplification product for 10 different genes, and HPRT1 was used as a reference gene for the ΔΔCT calculation [(Target – reference)Preamp – (Target – reference)cDNA]. Average ΔΔCT values were plotted for each cell number with Cells-to-CT lysates (A) and purified RNA (B).

Gene Expression Analysis from Preamplified cDNA without RNA Isolation

Traditionally, the first step in gene expression experiments is isolation of RNA. Even using the quickest and simplest techniques, RNA isolation requires significant hands-on manipulation and time. Some techniques also require cumbersome heating or centrifugation steps. These sample preparation steps are avoided with the TaqMan PreAmp Cells-to-CT Kit by use of an innovative set of lysis reagents for cultured mammalian cells.

The lysis protocol is extremely fast (7 minutes) and requires no heating, centrifugation, or sample transfers. The cultured mammalian cells are lysed while endogenous RNA is protected and genomic DNA is degraded. The RNA in the lysate serves as an effective template for real-time RT-PCR without the need for purification. Cells-to-CT lysates can be efficiently reverse transcribed using the included Cells-to-CT 2X RT Buffer and 20X RT Enzyme Mix. The convenience of the 2X RT Buffer, which contains dNTPs and oligonucleotides, further simplifies the system. The TaqMan® PreAmp Master Mix components in the kit then allow unbiased, simultaneous preamplification of up to 100 templates with TaqMan Gene Expression Assays. The TaqMan PreAmp Cells-to-CT™ protocol enables >60-fold more real-time PCRs than would be possible without the preamplification step, allowing substantially more information to be gathered from each sample collected.

The entire workflow (Figure 1) was tested using 100 assays in a single preamplification reaction and compared to preamplified purified RNA (Figure 2). Further, use of the TaqMan PreAmp Cells-to-CT Kit with low cell numbers (as few as 10 cells) generated more consistent and sensitive data than experiments using purified RNA (Figure 3).

Equivalent Expression Profile from Cell Lysates and Purified RNA

HeLa cells were washed with PBS and lysed using the Cells-to-CT reagents, or RNA was purified using the Supplier Q column-based method. The Cells-to-CT cell lysate method requires only 3 pipetting steps, while isolating RNA using the Supplier Q Kit requires at least 8 steps and removal of wash solutions from each collection tube after centrifugation. Less sample handling and hands-on time is a major advantage of the TaqMan PreAmp Cells-to-CT Kit, because there are fewer opportunities for mixing up samples and introducing variability during sample preparation.

After sample preparation was complete, samples were reverse transcribed using the Cells-to-CT RT reagents. The capacity, efficiency, and rigorous linearity of the RT reagents enable synthesis of maximal yields of cDNA from a wide range of sample amounts. After preamplification for 14 cycles with 100 TaqMan Gene Expression Assays, the product was diluted 1:20 with Tris:EDTA (TE) Buffer. Finally, real-time PCRs were assembled containing 25% diluted preamplification product or 25% RT reaction product (non-preamplified control) and one of the 100 TaqMan Gene Expression Assays used for preamplification. The real-time reactions were run with the supplied Applied Biosystems TaqMan Gene Expression Master Mix on a 7900HT Fast Real-Time PCR System under universal cycling conditions.

Figure 2 shows a comparison of the gene expression data obtained from Cells-to-CT lysates or purified RNA with (Panel B) or without (Panel A) preamplification. The correlation between Cells-to-CT lysates and purified RNA both before and after preamplification is excellent, with generally improved sensitivity when Cells-to-CT lysates are used. Further, the data show that the expression profile was not biased when using preamplified or non-preamplified lysates.

To further characterize the benefits of using the TaqMan PreAmp Cells-to-CT Kit with low cell numbers, HeLa cells were serially diluted from 100,000 to 10 cells per sample. RNA was purified using the Supplier Q column-based method or cells were lysed using the TaqMan PreAmp Cells-to-CT Kit. Samples were reverse transcribed, preamplified, and analyzed by real-time PCR for 10 genes as described above. To accurately express preamplification uniformity, a ΔΔCT calculation was performed for preamplified cDNA versus non-preamplified cDNA with HRPT1 as the reference gene, and then averaged over all analyzed genes. If the expression profile is unchanged after preamplification, the ΔΔCT value would be near zero. For each cell number, the Cells-to-CT lysate data were more consistent and closer to the ideal value of zero than the purified RNA data (Figure 3). This is especially apparent at 10 cells where column-based RNA purification results in variability and high sample loss, but lysis with Cells-to-CT reagents shows excellent performance.

The TaqMan PreAmp Cells-to-CT Kit allows expansion of precious starting material for real-time PCR in a simple and convenient workflow for researchers handling from one to ≥384 samples at a time. The kit is available in a 40-reaction size, enabling 40 lysis reactions, 40 RT reactions (and 40 minus RT controls), 40 preamplification reactions, and 500 real-time PCRs (20 µL each). TaqMan Gene Expression Assays (i.e., the primers and probe) are sold separately. The TaqMan PreAmp Cells-to-CT Kit is also fully compatible with the TaqMan Cells-to-CT Control Kit, which may be used to detect RT and real-time PCR inhibitors using the exogenous XenoRNA™ Control. This can then be used to determine appropriate cell numbers for specific applications. See sidebar for more information.

TaqMan® Cells-to-CT™ Control Kit

The new TaqMan Cells-to-CT Control Kit provides a control to detect inhibitors of RT-PCR and validation of sample input. This kit includes XenoRNA™ Control, a synthetic RNA transcript with a unique sequence that lacks homology to current, annotated biological sequences, and two TaqMan Gene Expression Assays—for XenoRNA Control and ß-actin, a housekeeping gene with moderately abundant expression levels in many cell types.

The XenoRNA Control can be added to the Stop Solution, which is then added to samples at the last step of the cell lysis. This provides a constant amount of control target in each sample, which can be amplified using the XenoRNA TaqMan Gene Expression Assay to confirm that samples can support RT-PCR.

ß-actin amplification results can be used as endogenous controls for normalization, as well as to ensure that a sufficient number of cells was included in the lysis reaction—this can be important for experiments with fewer than ~100 cells and to monitor the effectiveness of cell lysis.

XenoRNA Control and ß-actin can be used together to identify the maximum number of cells that can be used per Cells-to-CT lysis reaction. For these investigations, we recommend serially diluting cells and preparing a lysate that contains the XenoRNA Control. Then, use each lysate in parallel RT-PCRs with the TaqMan Assays for both XenoRNA Control and ß-actin. Depending on the experimental goals, it may also be useful to include a third RT-PCR targeting the gene of interest to evaluate the lower limits of cell input for target detection.